The success of gene therapy techniques depends largely on the ability to achieve a combination of stable chromosomal integration and high-level, regulated expression of transferred genes in a manner safe to humans. Many current techniques allow efficient transient transfection of cells in vitro and in vivo with large DNA fragments. However, subsequent chromosomal integration is very inefficient. To overcome low levels of integration, retroviral vectors, which integrate very efficiently in permissive cells, can be used.
While recombinant retroviral vectors allow for integration of a transgene into a host cell genome, most retroviruses can only transduce dividing cells, which limits their use for in vivo gene transfer to nonproliferating cells such as hepatocytes, myofibers, hematopoietic stem cells, and neurons. Non-dividing cells are the predominant, long-lived cell type in the body, and account for most desirable targets of gene transfer, including liver, muscle, and brain. Even protocols attempting the transduction of hematopoietic stem cells require demanding ex vivo procedures for triggering cell division in these cells prior to infection.
One way of overcoming this obstacle is to employ lentiviral vectors, in place of conventional retroviral vectors. Lentiviruses are complex retroviruses which, based on their higher level of complexity, can integrate into the genome of nonproliferating cells and modulate their life cycles, as in the course of latent infection. These viruses include HIV-1, HIV-2 and SIV. Like other retroviruses, lentiviruses possess gag, pol and env genes which are flanked by two long terminal repeat (LTR) sequences. Each of these genes encodes multiple proteins, initially expressed as one precursor polyprotein. The gag gene encodes the internal structural (matrix capsid and nucleocapsid) proteins. The pol gene encodes the RNA-directed DNA polymerase (reverse transcriptase, integrase and protease). The env gene encodes viral envelope glycoproteins and additionally contains a cis-acting element (RRE) responsible for nuclear export of viral RNA. The 5xe2x80x2 and 3xe2x80x2 LTRs serve to promote transcription and polyadenylation of the virion RNAs. The LTR contains all other cis-acting sequences necessary for viral replication. Adjacent to the 5xe2x80x2 LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient encapsidation of viral RNA into particles (the Psi site). If the sequences necessary for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, the result is a cis defect which prevents encapsidation of genomic RNA. However, the resulting mutant is still capable of directing the synthesis of all virion proteins. A comprehensive review of lentiviruses, such as HIV, is provided, for example, in Field""s Virology (Raven Publishers), eds. B. N. Fields et al., (copyright) 1996.
In addition to gag, pol, and env, lentiviruses, unlike other retroviruses, have several xe2x80x9caccessoryxe2x80x9d genes with regulatory or structural function. Specifically, HIV-1 possesses at least six such genes, including Vif, Vpr, Tat, Rev, Vpu and Nef. The closely related HIV-2 does not code for Vpu, but codes for another unrelated protein, Vpx, not found in HIV-1.
The Vpr gene encodes a 14 kD protein (96 amino acids) (Myers et al. (1993) Human Retroviruses and AIDS, Los Alamos National Laboratory, N.M.). The Vpr open reading frame is also present in most HIV-2 and SIV isolates. Amino acid comparison between HIV-2 Vpr and Vpx shows regions of high homology suggesting that Vpx may have arisen by duplication of the Vpr gene (Myers et al. (1993), supra.). Vpr and Vpx are present in mature viral particles in multiple copies, and have been shown to bind to the p6 protein which is part of the gag-encoded precursor polyprotein involved in viral assembly (WO 96/07741; WO 96/32494). Thus, incorporation of Vpr and Vpx into viral particles occurs by way of interaction with p6 (Lavallee et al. (1994) J. Virol. 68: 1926-1934; and Wu et al. (1994) J. Virol. 68:6161). It has been further shown that Vpr associates, in particular, with the carboxy-terminal region of p6. The precise role of Vpr and Vpx is yet to be clearly determined, however the data to date suggests that these proteins have a role in the stage of early infection. It has also been shown that Vpr and Vpx, expressed in trans with respect to the HIV genome, can be used to target heterologous proteins to HIV virus (WO 96/07741; WO 96/32494). A description of the structure and function of Vpr and Vpx, including the full-length nucleotide and amino acid sequences of these proteins and their binding domains are also provided in WO 96/07741, as well as in Zhao et al. (1994) J. Biol. Chem. 269(22):1577 (Vpr); Mahalingham et al. 91995) Virology 207:297 (Vpr); and Hu et al. (1989) Virology 173:624) (Vpx). Other relevant references relating to Vpr include, for example, Kondo et al. (1995) J. Virol. 69:2759; Lavallee et al. (1994) J. Virol. 68:1926; and Levy et al. (1993) Cell 72:541. Other relevant references relating to Vpx include, for example, Wu et al. (1994) J. Virol. 68:6161. All of the aforementioned publication are incorporated by reference herein.
In view of the advantages associated with retroviral vectors in gene therapy, particularly lentiviruses which are capable of infecting non-dividing cells, improved methods for generating pure stocks of recombinant virus, free of replication-competent helper virus, would be of great value in the art. Recombinant retroviruses are generally produced by introducing a suitable proviral DNA vector into mammalian cells (xe2x80x9cpackaging cellsxe2x80x9d) that produce the necessary viral proteins for encapsidation of the desired recombinant RNA, but which lack the signal for packaging viral RNA ("psgr" sequence). Thus, while the required gag, pol, and env genes of the retrovirus are intact, there is no release of wild-type helper virus by these packaging lines. However, when the cells are transfected with a separate vector containing the "psgr" sequence required for packaging, wild-type retrovirus can arise by recombination (Mann et al. (1983) Cell 33:153). This is a major danger, particularly in the case of lentiviruses, such as HIV.
Current approaches to avoid the safety dangers associated with recombination leading to production of replication-competent helper virus include making additional mutations (e.g., LTR deletions) in the viral constructs used to create packaging lines, and separating the viral genes necessary for producing virions onto separate plasmids. For example, it has recently been shown that recombinant Moloney murine leukemia virus (MuLV), free of detectable helper-virus, can be produced by separating the gag and pol genes from the env gene in packaging cells (Markowitz et al. (1998) J. Virol. 62(4):1120). These packaging cells contained two separate plasmids collectively encoding the viral proteins necessary for virion production, reducing the likelihood that the recombination events necessary to produce intact retrovirus (i.e., between three plasmid vectors) would occur when cotransfected with a third vector containing the "psgr" packaging signal.
Additional methods for producing safer retroviral packaging cell lines, particularly lentiviral packaging cell lines, which generate recombinant retrovirus, yet do not themselves either yield detectable helper virus or transfer viral genes, would be of great value in human gene therapy.
The present invention provides novel packaging cell lines which produce recombinant retrovirus, free of detectable helper-virus. Retroviruses produced by the cell lines of the invention include lentiviruses, such as HIV, capable of transferring heterologous DNA to a wide range of non-dividing cells. Among other advantages, the packaging cells provide the benefit of increased safety when used in human gene therapy by virtually eliminating the possibility of molecular recombination leading to production of replication-competent helper virus.
In one embodiment, the invention provides a retroviral packaging cell line containing at least three separate expression vectors which collectively encode gag, pol, and env polyproteins and which, unlike other packaging cell lines, separate the gag and pol genes, at least in part, onto different vectors to reduce the likelihood of recombination with other retroviral vectors within the cell, leading to the production of replication-competent helper virus. The first vector, referred to as pgag{circumflex over ( )}pol, encodes the complete gag polyprotein (containing viral matrix, capsid and nucleocapsid proteins, such as p17, p24, p9 and p6) and, in certain embodiments, also encodes a portion of the pol polyprotein (containing viral polymerase proteins, such as protease, reverse transcriptase and integrase). In one embodiment, the portion of pol encoded along with gag in the first vector includes the protease (PR) protein. In most lentiviral genomes, PR is encoded by a region of pol which overlaps with gag (see FIG. 5). Therefore, in this embodiment, the first vector encodes gag and the portion of pol which overlaps with gag in the lentiviral (e.g., HIV) genome. Other overlapping or non-overlapping portions of pol can also be included with gag on the first vector. However, in another embodiment, gag and pol are completely separated so that the first vector encodes all of gag and no portion of pol.
The second vector, referred to as pVpr-RTIN, complements the first vector, pgag{circumflex over ( )}pol, by encoding the remaining portion(s) of the pol polyprotein not encoded by pgag{circumflex over ( )}pol. The pol polyprotein includes protease (PR), reverse transcriptase (RT) and integrase (IN). Thus, in the case where pgag{circumflex over ( )}pol encodes PR, the second vector preferably encodes RT and IN. In addition, the second vector encodes a targeting protein which targets the encoded pol proteins (e.g., RT and IN) to assembling virions at the inner face of the plasma membrane. Normally, pol is directed to assembling virions via gag since they are expressed together as one large gag-pol precursor polyprotein (e.g., Pr160gag-Pol). However, in the vectors of the present invention, gag and at least a portion of pol are separated onto different vectors. Thus, the invention employs an agent which targets the portion of pol encoded by the second vector to assembling virions. The targeting agent (e.g., protein or peptide) is preferably encoded in frame with the portion of pol, so that the vector is expressed as a single fusion protein.
Any suitable targeting agent which binds to a component of assembling retroviral virions (e.g., lentiviral gag proteins) can be encoded (e.g., along with a portion of pol) by the second vector. Suitable targeting agents include, for example, antibodies, antibody fragments, proteins and peptides. In one embodiment, the targeting protein is either Vpr or Vpx, including fragments or mutants thereof, which bind to p6 gag protein. Thus, in one embodiment, the second vector encodes a Vpr or Vpx fusion protein containing Vpr or Vpx (or peptides, mutants or variants thereof) and a portion of pol, where the portion of pol preferably includes RT and IN. Within this fusion construct, RT and IN are preferably preceded by a protease cleavage site so that they are cleaved and activated by PR once they become associated with assembling virions.
The third vector, referred to as pENV, encodes a viral env which provides one or more envelope proteins for viral particles encoded by the first and second vectors. In one embodiment, the viral env is from a lentivirus, such as HIV, SIV, FIV, EIV (e.g., gp120 and gp41). In another embodiment, the viral env is from VSV (e.g., VSV-G glycoprotein which pseudotypes the recombinant retroviral particles encoded by the first and second vectors). In yet another embodiment, the viral env is from a Type C retrovirus, such as MoMuLV, HaMuSV, MuMTV, GaLV, FLV and RSV.
The first, second and third vectors described above are cotransfected into suitable packaging cells, such as 293T human kidney cells, to produce novel packaging cell lines of the invention. When cotransfected with a fourth vector, which contains the necessary xcexa8 and LTR sequences for packaging of RNA into viral particles, the cells produce recombinant, helper-free retrovirus. Accordingly, in another embodiment, the invention provides a producer cell line containing a fourth vector (along with the first, second and third vectors), referred to as pxcexa8. The fourth vector comprises a retroviral packaging signal (xcexa8), preferably along with a selected transgene, flanked by long terminal repeat sequences (LTRs). Any of the first, second, third or fourth vectors also can contain an RNA export element, such as the HIV RRE, and/or a marker gene enabling the detection of positive cell transformants, as well as unwanted helper-virus.
In another embodiment, the invention provides a method of producing a packaging cell line capable of generating recombinant, helper-free retrovirus. The method involves transfecting a suitable host cell with a first vector encoding a retroviral gag polyprotein along with (in certain embodiments) a portion of a retroviral pol protein, a second vector encoding the remainder of the retroviral pol polyprotein not encoded by the first vector fused to a Vpr or a Vpx protein, and a third vector encoding a viral env protein, each as described above. Each one of the first, second or third vectors contains a promoter operably linked to a gene encoding the gag polyprotein, pol polyprotein, Vpr protein, Vpx protein or env protein. In one embodiment, the promoter is an inducible promoter allowing for selective expression of the gag polyprotein, pol polyprotein, Vpr protein, Vpx protein or env protein within the packaging cells.
In yet another embodiment, the invention provides a method of producing a recombinant retrovirus by co-transfecting a host cell with a first vector comprising a retroviral gag gene and (in certain embodiments) a portion of a retroviral pol gene, both operably linked to a promoter; a second vector comprising all or the remaining portion of the retroviral pol gene not contained within the first vector and a gene encoding all or a portion of a Vpr or a Vpx protein, the genes being operably linked to a promoter and expressed as a single fusion protein; a third vector comprising a viral env gene; and a fourth vector comprising a viral packaging signal, a viral long terminal repeat (LTR), and preferably a transgene, all as described above. Following cotransfection of the first, second, third and fourth vectors into suitable cells, recombinant retrovirus can be recovered from the cell culture medium.
Packaging cell lines of the invention, and recombinant retroviruses (e.g., HIV and SIV) produced from these cell lines, can be used to deliver heterologous nucleic acids (e.g., therapeutic transgenes) to dividing and non-dividing cells in a safe and efficient manner. For example, they can be used to transform target cells with a desired DNA in vitro. Additionally, they can be used in vivo to deliver therapeutic genes to cells (e.g., in methods of human gene therapy) without the danger of recombination leading to the producing replication-competent helper-virus.